Anti-ABL1 antibody [EPR23406-32]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ABL1 antibody [EPR23406-32] (AB254341)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling ABL1 with ab254341 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-ABL1 antibody [EPR23406-32] (AB254341)

False colour image of Western blot : Anti-ABL1 antibody [EPR23406-32] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab254341 was shown to bind specifically to ABL1. A band was observed at 123 kDa in wild-type HeLa cell lysates with no signal observed at this size in ABL1 knockout cell line ab277152 (knockout cell lysate ab277194). To generate this image, wild-type and ABL1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ABL1 antibody [EPR23406-32] (ab254341) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 3:

K562 cell lysate at 20 µg

Predicted band size: 122 kDa

Observed band size: 130 kDa

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• WB

Lab

Western blot - Anti-ABL1 antibody [EPR23406-32] (AB254341)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Fresh lysates are recommended for loading to minimize target degradation.

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-ABL1 antibody [EPR23406-32] (ab254341) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

Daudi (human burkitts lymphoma lymphoblast) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 122 kDa

Observed band size: 135 kDa

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• WB

Lab

Western blot - Anti-ABL1 antibody [EPR23406-32] (AB254341)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-ABL1 antibody [EPR23406-32] (ab254341) at 1/1000 dilution

All lanes:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 122 kDa

Observed band size: 135 kDa,210 kDa

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• WB

Lab

Western blot - Anti-ABL1 antibody [EPR23406-32] (AB254341)

Lanes 1 - 3 : Merged signal (red and green). Green - ab254341 observed at 130 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab254341 was shown to react with ABL1 in wild-type HeLa cells in western blot. The bands observed in ABL1 knockout cell line ab265612 (ABL1 knockout cell lysate ab263077) below 130 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. HeLa wild-type and ABL1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1 % Tween^®) before incubation with ab254341 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 : 1000 dilution and a 1 : 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 : 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ABL1 antibody [EPR23406-32] (ab254341) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ABL1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ABL1 knockout HeLa cell line (<a href='/ko/products/cell-lines/human-abl1-knockout-hela-cell-line-ab265612'>ab265612</a>)

Lane 3:

K562 cell lysate at 20 µg

Predicted band size: 122 kDa

Observed band size: 130 kDa

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